Expression of cyclins A, D2 and D3 in individual normal mitogen stimulated lymphocytes and in MOLT-4 leukemic cells analyzed by multiparameter flow cytometry.

Cyclins are regulatory subunits of the cyclin dependent kinases (CDKs), the enzymes that drive the cell through the respective phases and check-points of the cell cycle. The expression of cyclins in non-tumor cells, regulated by timely induction of their synthesis and proteolysis, is scheduled, occurring at discrete periods of the cell cycle. Using multiparameter flow cytometry we have recently observed that expression of cyclins B1 and E in individual normal lymphocytes mitogenically stimulated by phytohemagglutinin (PHA) and lymphocytic leukemic MOLT-4 cells was similar, restricted to particular phases of the cycle: cyclin B1 was detected only in G2+M- and cyclin E in late G1 and early S-phase cells. In the present study we have measured the expression of cyclins A, D2 and D3 in these cells. The presence of cyclin A was restricted to late S and G2 phases, both in the case of lymphocytes and of MOLT-4 cells. Over 95% of the non-stimulated lymphocytes were both cyclin D2 and D3 negative. Mitogenic stimulation with PHA-induced expression of cyclins D2 and D3 in over 50% cells, which corresponds to the percentage of cells that respond to this mitogen in cultures. Expression of these proteins peaked between 8 and 24 h after addition of PHA, and then decreased at the time of cell entrance to S. During exponential growth (48-72 h after stimulation with PHA) expression of the D-type cyclins was diminished: only between 5-10% of the lymphocytes had levels of cyclin D3 as high as G1 cells between 8-24 h after PHA stimulation. Populations of proliferating lymphocytes and MOLT-4 cells were very heterogeneous in terms of expression of D-type cyclins by individual cells. While expression of cyclin D2 in exponentially growing MOLT-4 cells was similar to that of proliferating lymphocytes, the percent of cells expressing cyclin D3 as well as the degree of expression, was higher in MOLT-4 cells, regardless of the phase of the cycle. These results, with our earlier observations of the untimely expression of cyclins B1 and E in several other tumor lines, suggest that altered expression of cyclins may be a frequent feature of malignancy.